The site-specific incorporation of bio-orthogonal groups via genetic code expansion provides a powerful general strategy for site specifically labelling proteins with any probe. However, the slow reactivity of the bio-orthogonal functional groups that can be genetically encoded, and/or their need for photoactivation, has limited this strategy's utility.
The rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chemical biologists; enzyme mediated labeling approaches may be rapid, but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many ‘bio-orthogonal’ reactions for which a component can be genetically encoded are too slow to effect the quantitative and site specific labeling of proteins on a time-scale that is useful to study many biological processes.
There is a pressing need for general methods to site-specifically label proteins, in diverse contexts, with user-defined probes.
Inverse electron demand Diels-Alder reactions involving tetrazines have emerged as an important class of rapid bio-orthogonal reactions. The rates reported for some of these reactions are very fast.
Yu et al 2012 (Angew. Chem. Int. Ed. Volume 51, pages 10600-10604) disclose Genetically Encoded Cyclopropene Directs Rapid, Photoclick Chemistry Mediated Protein Labelling in Mammalian Cells. The authors report the synthesis of a stable cyclopropene amino acid, the characterisation of its reactivity in a photo induced cycloaddition reaction with two tetrazoles, its site-specific incorporation into proteins both in E. coli and in mammalian cells, and its use in directing bioothogonal labelling of proteins both in vitro and in vivo. In order to incorporate their cyclopropene containing amino acid into proteins, the authors had to evolve an orthogonal tRNA/tRNA synthetase pair that selectively charges their cyclopropene lysine amino acid in response to a TAG amber codon. This required a synthetase library to be constructed, five positions within that synthetase to be randomised, together with at least five rounds of positive and negative selection screening. It is a drawback of this work that it relies on the specific mutant synthetase produced. In joining their tetrazole compounds to the cyclopropene moiety in their modified amino acids, Yu et al use photo activation. Photo activation is carried out at either 302 nano metres or 365 nano metres. The requirement for photo activation in joining tetrazoles to the amino acid of Yu et al is a drawback in the art. This is a laborious extra step in the conjugation chemistry. UV is also damaging to cells and so is disadvantageous in the in vivo/cellular setting.
Kamber et al disclose Isomeric Cyclopropenes Exhibiting Unique Bioorthogonal Reactivities (2013 JACS Volume 135, pages 13680-13683). The authors discuss two reactions that can be used to tag biomolecules in complex environments: the inverse electron demand Diels-Alder reaction of tetrazines with 1,3-disubstituted cyclopropenes, and the 1,3-dipolar cycloaddition of nitrile imines with 3,3-disubstituted cyclopropenes. The authors discuss various chemical reaction schemes used to generate stable cyclo adducts. None of the molecules discussed by Kamber et al are amino acids. There is no reason to imagine that the compounds as described could be incorporated into amino acids. Even if any such incorporation was attempted, there is absolutely no suggestion or guidance which might allow such compounds to be incorporated into polypeptides. No schemes for synthesis of amino acids comprising any of the chemical groups described are presented by Kamber et al. There are no biochemical tools for incorporation into proteins mentioned anywhere in this document. Kamber et al are solely concerned with examining the substitution pattern on the cyclopropene, one such pattern allowing reactions with tetrazines and one such pattern not being permissive of reactions with tetrazines.
The present invention seeks to overcome problem(s) associated with the prior art.